The present invention generally relates to the field of imaging. In particular, the present invention provides scanning systems and methods for high speed imaging of a sample containing labeled materials, and particularly for scanning arrays of polymer sequences, e.g., oligonucleotide arrays.
Methods and systems for imaging samples containing labeled markers such as confocal microscopes are commercially available. Confocal microscopes generally employ a pinhole that is confocal with the illuminated spot on the specimen to reject light that is not reflected or emitted from objects in the focal plane. This rejection of out-of-focus light enables the microscope to collect and combine a series of optical slices at different focus positions to generate a two or three dimensional representation of the specimen.
Some scanning microscopes employ radiation direction systems, such as galvanometers that include servo-mounted mirrors, to rapidly scan a laser spot across a substrate. Although these microscopes have relatively high scan rates (e.g., on the order of about 30 lines/second or greater), they generally do not achieve both the resolution and field of view that is necessary for some applications, such as imaging an array of sequenced materials on a substrate. In fact, a galvanometer-based confocal microscope""s field of view is generally proportional to its resolution. For example, a typical 40xc3x97 microscope objective, which has a 0.25 xcexcm resolution, has a field size of only about 500 xcexcm. Thus, conventional galvanometer-based confocal microscopes are inadequate for applications requiring both high resolution and a large field of view.
Scanning confocal microscope systems, such as those discussed in U.S. Pat. No. 5,143,854 (Pirrung, et al.), PCT WO 92/10092, and U.S. patent application Ser. No. 08/195,889 (Attorney Docket Number 16528X-006000), incorporated herein by reference for all purposes, are also known. These scanning systems include an optical train which directs a monochromatic or polychromatic light source to about a 5 micron (xcexcm) diameter spot at its focal plane. In some cases, a photon counter detects the emission from the device in response to the light. The data collected by the photon counter represents one pixel or data point of the image. Thereafter, the light scans another pixel as a translation stage moves the device to a subsequent position.
As disclosed, these scanning confocal microscope systems provide high resolution by using an appropriate objective lens and large field of view by using appropriate translation stages. These translation stage-based confocal microscopes, however, obtain high resolution and field of view by sacrificing system throughput. As an example, an array of sequenced material using the pioneering fabrication techniques, such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.) and U.S. patent application Ser. No. 08/143,312, incorporated herein by reference for all purposes, may have a density of about 105 sequences. Assuming that 36 pixels are required for each sequence, the image can take over at least 10 minutes to acquire.
From the above, it is apparent that improved methods and systems for imaging a sample are desired.
The present invention provides systems, methods and apparatus for detecting marked regions on substrate surfaces. In particular, the present invention provides methods and apparatus for scanning a substrate to obtain an image with high sensitivity and resolution at a high speed. The confocal scanning microscopes of the present invention combine the high scan rate of galvanometer based scanning microscopes with a sufficiently high resolution, sensitivity and a large enough field of view for imaging high density arrays of materials, such as those found in the fields of combinatorial chemistry and genetic analysis.
In one aspect, the present invention provides a system for detecting marked regions on a surface of a substrate, which comprises an excitation radiation source, and focusing optics for focusing the excitation radiation to regions on the surface of the substrate. A radiation direction system is also included for linearly scanning the focused excitation radiation across the surface of the substrate. A detector is positioned for detecting an emission from the substrate surface in response to the excitation radiation, and a data acquisition system records the amount of detected emission as a function of a position on the surface of the substrate from which the emission was detected.
In one embodiment, the focusing optics comprises an objective lens having a ratio of scanning field diameter to focused spot diameter of greater than 2000, preferably greater than 3000 and more preferably greater than 4000. Thus, the microscope is, for example, capable of focusing a laser beam to a spot having a diameter of about 3 microns at any point within a flat field having a length of about 14 mm. In addition, the objective lens has at least a 0.2 numerical aperture, preferably a 0.25 numerical aperture, which provides sufficient sensitivity to detect fluorescently marked regions on the substrate.
The radiation direction system preferably comprises a galvanometer mirror that scans the excitation radiation across the surface of the substrate. The objective lens has an external entrance pupil preferably located at or near the galvanometer mirror""s pivot location. The mirror usually oscillates at a frequency of at least 7.5 Hz, preferably at least 20 Hz and more preferably at least 30 Hz. In this manner, the laser spot can usually be scanned across the substrate at velocities of about 5 image lines/second, preferably at least 10 images lines/second, and more preferably at least about 30 image lines/second. This allows the microscope to rapidly scan high density substrates, such as the polymer array substrates disclosed by Pirrung. It should be noted that the mirror may scan unidirectionally (e.g., with a sawtooth wave) or bidirectionally (e.g., with a symmetric triangle wave). In the latter case, the galvanometer frequency would generally be about half of the data acquisition speed in image lines/second. Accordingly, the frequency of the galvanometer in the latter case may be lower than 7.5 Hz in order to scan 5 image lines/second.
In another embodiment, the present invention also provides a system for detecting fluorescent regions on a surface of a substrate, which comprises an excitation radiation source, and first focusing optics for focusing the excitation radiation on the surface of the substrate in a focused spot having a diameter no greater than 10 xcexcm, preferably less than 5 xcexcm and more preferably about 31 xcexcm. An oscillating or reciprocating radiation direction system scans the spot linearly across the surface of the substrate, with a focused travel distance of at least 10 mm and preferably about 14 mm. In one embodiment, an optical train separates fluorescence emitted from the surface of the substrate from the excitation radiation reflected from the surface. An autofocus system may also be included for automatically placing the surface of the substrate in a focal plane of the focusing optics.
The present invention also provides methods of scanning substrates using the above systems. For example, in one aspect, the invention provides a method of scanning a polymer array having a plurality of different polymer sequences, each of the different polymer sequences being immobilized on a surface of a substrate in a different known location, to identify which polymer sequence on the array is bound by a fluorescent target molecule. The method comprises focusing an excitation radiation source upon the surface of the substrate, and scanning the excitation radiation across the surface of the substrate at a speed of at least 5 image lines/second. Fluorescent emissions are collected from the surface of the substrate in response to the excitation radiation. These fluorescent emissions are recorded as a function of a position on the surface of the substrate. The position on the surface indicates the polymer sequence on the array that is bound by the fluorescent target molecule.
A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.